The present invention relates to synthetic reagents or substrates which are used for the quantitative determination of proteolytic enzymes. More particularly, the invention relates to synthetic thioesters which are useful as a reagent for the quantitative determination of proteolytic enzyme of class E.C. 3.4.21, which split peptide chains on the carboxyl side of arginine as well as lysine in human and mammal body fluids as well as in vegetable and animal cell extracts and in glandular venoms of cold-blooded animals such as snakes.
Classical substrates for trypsin, thrombin and related enzymes have involved both esters such as .alpha.-N-tosyl-L-arginine methyl ester and .alpha.-N-tosyl-L-lysine methyl ester [G. W. Schwert et al., J. Biol. Chem., 172 (1948) 221; Sherry, S. and Troll, W., J. Biol. Chem., 208 (1954) 95; Elmore, D. T. and Curragh, Z. F. Biochem. J., 86 (1963)98]as well as amides such as .alpha.-N-benzoyl-DL-arginine-p-nitroanilide, L-lysine-p-nitroanilide, .alpha.-N-benzoyl-DL-arginine-2-naphthylamide and other di, tri and higher order arginine and lysine peptides with chromogenic amide leaving groups [B. F. Erlanger et al., Arch. Bioch. Biop. 96 (1961) 271; A. Redidel and E. Wunsch, Z. Physiol. Chem. 316 (1959) 1959; R. E. Plapinger et al., J. Org. Chem. 30 (1965) 1781; L. Svendsen et al., Thrombosis Res. 1 (1972) 267].
The use of ester substrates for this class of enzyme have been limited by the cumbersome assay procedures such as pH titration or detection of the small change in absorbance of the above products in the UV region of the spectra. The introduction of the amide chromogenic substrates based on p-nitroaniline or similar leaving groups have offered the advantage of an assay in the visible region of the spectra with by-products which have high extinction coefficients enabling more sensitive enzyme determinations to be made. The simple chromogenic substrate based only upon arginine or lysine amides have proved to be extremely poor substrates relative to the analogous esters [Erlanger et al., Arch. Bioch. Biop. 95 (1961) 271-8]. The advantage of extending the amino terminal end of either arginine or lysine p-nitroanilide substrates has been well documented and results in much improved substrate behavior, especially as determined by typical Michaelis-Menten kinetic parameters [Thrombosis Res 1 (1972) 267-78; U.S. Pat. No. 3,884,896, U.S. Pat. No. 4,061,625]. The difficulty in synthesizing these tri and tetra peptide materials has resulted in a high cost of the substrates and at the same time certain disadvantages such as limited solubility and inhibition of the enzymes by the cleaved product p-nitroaniline.
Farmer and Hageman, the Journal of Biological Chemistry, 250, 7366 (1975) describe the use of N-benzoyoyl-L-tyrosine thiobenzyl ester as a protease substrate detected by further reaction with 5,5'-dithiobis [2-nitrobenzoic acid].
Substrates of the present invention are particularly advantageous in that they have selective activities toward trypsin and they have excellent shelf life.
p-guanidino-L-phenylalanine methyl ester is known as a substrate for trypsin and as an inhibitor of thrombin, Klausner et al., Biochem. J. 169, 157-167 (1978). Compounds of the present invention provide reagents which are selective in their activity, have stable shelf life and the product of enzyme hydrolysis is readily converted to a colorimetric reagent.